Plant proteins with antifungal activity are known. A chitinase purified from bean causes inhibition of the growth of the fungus Trichoderma viride (Schlumbaum et al., (1986), Nature 324, 365-367).
A pea chitinase with a growth inhibitory effect on Trichoderma viride in agar plate tests is described by Mauch et al., (1988, Plant Physiol. 88, 936-942). This enzyme, however, only has a limited effect on for instance the ascomycete Cladosporium cucumerinum, and no effect on the growth of inter alia the Oomycetes Phytophthora cactorum, Pythium aphanidermatum, and Pythium ultimum. Hence, an important disadvantage of this enzyme is its limited working range. In a similar test it was established that .beta.-1,3-glucanase has a growth inhibitory effect on Fusarium solani f.sp. pisi.
A preparation with a hydrolytic effect on isolated cell walls of Verticillium alboatrum, comprising a combination of a purified endo-.beta.-1,3-glucanase from tomato and an exo-.beta.-1,3-glucanase from fungal origin is described by Young & Pegg (1982, Physiol. Plant Pathol. 21, 411-423). Both enzymes had no effect on their own.
Several thionines, inter alia from leaves of barley, maize, wheat, rye, and several dicotyledonous plants, showing a significant antifungal effect in in vitro tests, are described by Bohlmann, H. et al., (1988, EMBO J. 7, 1559-1565).
Furthermore, plant proteins with an enhancing effect on the fungus-inhibitory activity of antibiotics are described in International Patent Application PCT/US88/03420. These plant proteins are generally designated as Synergistic Antifungal Proteins or SAFPs. SAFPs are used in combination with polyoxines and nikkomycines, that are active on their own against phytopathogenic fungi; in combination with SAFPs improvements of the effectivity can be achieved in the order of 10 to 100. SAFPs have no antifungal effect on their own.
In plants, the synthesis of chitinases and glucanases, as well as a large number of different so-called pathogenesis-related (PR-) proteins, is known to be accompanied by a phenomenon known as the hypersensitive response, which is inter alia triggered by an incompatible plant pathogen. This hypersensitive response eventually results in resistance of the plant against a broad range of pathogens. Similarly, the synthesis of PR-proteins can be induced by a number of biotic and abiotic factors, such as fragments of fungal cell walls, chemical inducers, such as salicylate and the like, which also results in a broad pathogen-resistance of the plant. This resistance obtained through induction either by an incompatible pathogen or a biotic or abiotic factor, or chemical substance, is called `induced resistance`. Although still very much has to be learned about induced resistance and the role of these PR-proteins, some classification has been done. In tobacco, it seems that at least 5 classes of PR-proteins are induced upon treatment with tobacco mosaic virus (TMV). This classification is based on features such as molecular weight, serological relationship, amino-acid sequence homology, and if known, enzymatic activity. Within these classes a division can be made into intracellular and extracellular proteins, which except for their cellular localisation in the plant, correspond to each other with respect to the features just mentioned (vide for overview, Bol J. F. et al., 1990, Annu. Rev. Phytopathol. 28, 113-138.) Since these proteins are believed to be somehow involved in pathogen resistance, a great deal of effort is put into identification of potent antipathogenic proteins within the family of PR-proteins.
Upto the present, the approach for the screening and isolation of antifungal proteins is the screening of PR-proteins with already known properties, such as molecular weight, pI, or enzymatic activity. This especially holds for the chitinases and .beta.-1,3-glucanases, the substrates of which occur in the cell walls or integuments of most pathogens and/or pests. One disadvantage of this approach is that there seems to be no or little correlation between enzymatic (i.e. chitinase and glucanase) activity and antifungal effect, resulting in the often tedious isolation of proteins which turn out to have no significant antifungal effect. The second disadvantage is the even greater difficulty of isolating PR-proteins of which no activity or function is known, which is the case for the majority of the PR-proteins.
Therefore, there is a need for a more effective and reliable method to obtain proteins with a significant antipathogenic effect against a selected pathogen.